Erk1/2 activation is mainly induced by GPVI but not α2 ligation. (A,C) MKs were kept in suspension in BSA-coated dishes, allowed to adhere to CVX, the high-affinity substrate for GPVI or GFOGER peptide, the specific ligand of alpha2 integrin for different times. (B) MKs preincubated with the function-blocking anti-GPIbα (LJ-Ib1; lanes 2, 4, and 6) or the isotype-matched control (IgG1; lanes 1, 3, and 5) were kept in suspension (lanes 1-2) or allowed to attach to CVX substrate for 10 minutes (lanes 3-4) or 2 hours (lanes 5-6) in the presence of the antibodies (100 μg/mL). Suspended or adherent cells were lysed and subjected to Western blot analysis using an anti-phospho-Erk1/2 (P-Erk) antibody as indicated in “Materials and methods.” Equal loading was assayed by labeling with an anti-Erk antibody (Total Erk). Note that Erk phosphorylation induced by CVX was not affected by GPIbα blocking antibody (LJ-Ib1). The graphs illustrate densitometric quantitation of the ratio of phospho-Erk to total Erk in each condition. The mean ± SD of 3 independent experiments is shown.